Single-cell force spectroscopy of pili-mediated adhesion.

Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive single-cell force spectroscopy (SCFS), we quantify the adhesion forces between individual bacteria and biotic (mucin, intestinal cells) or abiotic (hydrophobic monolayers) surfaces. On hydrophobic surfaces, bacterial pili strengthen adhesion through remarkable nanospring properties, which - presumably - enable the bacteria to resist high shear forces under physiological conditions. On mucin, nanosprings are more frequent and adhesion forces larger, reflecting the influence of specific pili-mucin bonds. Interestingly, these mechanical responses are no longer observed on human intestinal Caco-2 cells. Rather, force curves exhibit constant force plateaus with extended ruptures reflecting the extraction of membrane nanotethers. These single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces (nanosprings, nanotethers), and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells (probiotics, pathogens).

Adhesion and nanomechanics of pili from the probiotic Lactobacillus rhamnosus GG.

Knowledge of the mechanisms by which bacterial pili adhere to host cells and withstand external forces is critical to our understanding of their functional roles and offers exciting avenues in biomedicine for controlling the adhesion of bacterial pathogens and probiotics. While much progress has been made in the nanoscale characterization of pili from Gram-negative bacteria, the adhesive and mechanical properties of Gram-positive bacterial pili remain largely unknown. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of pili from the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG (LGG). First, we show that SpaC, the key adhesion protein of the LGG pilus, is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Homophilic and heterophilic interactions display similar binding strengths and dissociation rates. Next, pulling experiments on living bacteria demonstrate that LGG pili exhibit two unique mechanical responses, that is, zipper-like adhesion involving multiple SpaC molecules distributed along the pilus length and nanospring properties enabling pili to resist high force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stresses in the natural environment. The single-molecule experiments presented here may help us to design molecules capable of promoting or inhibiting bacterial-host interactions.

Towards a nanoscale view of lactic acid bacteria.

Probiotic bacteria have a strong potential in biomedicine owing to their ability to induce various beneficial health effects. Bacterial cell surface constituents play a key role in establishing tight interactions between probiotics and their host. Yet, little is known about the spatial organization and biophysical properties of the individual molecules. In this paper, we discuss how we have been using atomic force microscopy imaging and force spectroscopy to probe the nanoscale surface properties of gram-positive lactic acid bacteria, with an emphasis on probiotic strains. Topographic imaging has enabled us to visualize bacterial cell surface structures (peptidoglycan, teichoic acids, pili, polysaccharides) under physiological conditions and with unprecedented resolution. In parallel, single-molecule force spectroscopy has been used to localize and force probe single cell surface constituents, providing novel insights into their spatial distribution and molecular elasticity.

Deciphering the nanometer-scale organization and assembly of Lactobacillus rhamnosus GG pili using atomic force microscopy.

In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.

Morphological spectrum of gastrointestinal tuberculosis.

INTRODUCTION: Gastrointestinal tuberculosis (GITB) is a great mimicker and it is often difficult to distinguish GITB from other inflammatory lesions of the intestine. AIM: This study was carried out with the objective of analysing the entire morphological spectrum of GITB. METHODS: A total of 110 diagnosed cases of GITB were included in the study. The diagnosis was based on the presence of acid-fast bacilli (AFB) on histology, caseating or non-caseating epithelioid cell granulomas (ECGs), evidence of tuberculosis at other extraintestinal sites, and all of these along with a complete response to anti-tuberculous treatment (ATT). RESULTS: The mean age was 30.9 years with M:F ratio of 1:1. On gross examination, apart from typical tuberculous lesions in the form of transverse ulcers, strictures, hyperplastic lesions and serosal tubercles, intestinal perforation (32.6%) was seen with higher frequency and ischemic bowel was also identified (7.3%). Varied morphological patterns of ECGs in the form of caseating, non-caseating, confluent, discrete and even suppurative granulomas were identified on histopathology. An important finding was the co-existence of different types of granulomas within the same case. In a significant number of cases (44.5%) granulomas were seen in a submucosal location. The predominant type of inflammation seen in the lamina propria was lymphoplasmacytic in 85.5% cases. CONCLUSION: Pathologists should be aware of the entire spectrum of gross and histopathological features of GITB, so as to avoid misdiagnosis.